All this may have an impact on LSJL but I’m not sure what it is yet.  However, nerves are something that you can feel.  However, different nerves have different effects so it’d be hard to gauge what the effect is.  But as alluded to in the study reduced pain sensitivity is a sign of reduced substance P and SNF.  So if you have reduced pain sensitivity due to LSJL you may have less SP and SNF.  The reduced mechanical stability may be beneficial to neo-growth plates also.  However, mesenchymal stem cells and chondrocytes stained heavily for SP so enhanced pain sensation may be more indicative of the success of LSJL.

Absence of substance P and the sympathetic nervous system impact on bone structure and chondrocyte differentiation in an adult model of endochondral ossification.

substance P pdf<-Read the full study.

“Sensory and sympathetic nerve fibers innervate bone and epiphyseal growth plate. The role of neuronal signals for proper endochondral ossification during skeletal growth is mostly unknown. Here, we investigated the impact of absence of sensory neurotransmitter substance P (SP) and removal of sympathetic nerve fibers (SNF) on callus differentiation, a model for endochondral ossification in adult animals, and on bone formation.
In order to generate callus, tibia fractures were set in the left hind leg of wild type (WT), tachykinin 1-deficient (Tac1-/-) mice (no SP) and animals without sympathetic nerve fibers. Locomotion was tested in healthy animals and touch sensibility was determined early after fracture. Callus tissue was prepared for immunofluorescence staining for SP, neurokinin1-receptor (NK1R), tyrosine-hydroxylase (TH) and adrenergic receptors α1, α2 and β2. At the fracture site, osteoclasts were stained for TRAP, osteoblasts were stained for RUNX2 and histomorphometric analysis of callus tissue composition was performed. Primary murine bone marrow derived macrophages (BMM), osteoclasts and osteoblasts were tested for differentiation, activity, proliferation and apoptosis in vitro. Femoral fractures were set in the left hind leg of all three groups for mechanical testing and μCT-analysis.
Callus cells stained positive for SP, NK1R, α1d- and α2b adrenoceptors and remained β2- adrenoceptor and TH-negative. Absence of SP and SNF did not change general locomotion but reduces touch sensitivity after fracture. In mice without SNF, we detected more mesenchymal callus tissue and less cartilaginous tissue 5days after fracture{so more sympathetic nerve fibers are pro-differentiation?}. At day 13 past fracture, we observed a decrease of the area covered by hypertrophic chondrocytes in Tac1-/- mice and mice without SNF, a lower number of osteoblasts in Tac1-/- mice and an increase of osteoclasts in mineralized callus tissue in mice without SNF. Apoptosis rate and activity of BMM, osteoclasts and osteoblasts isolated from Tac1-/- and sympathectomized mice were partly altered in vitro. Mechanical testing of fractured- and contralateral legs 21days after fracture, revealed an overall reduced mechanical bone quality in Tac1-/- mice and mice without SNF. μCT-analysis revealed clear structural alteration in contralateral and fractured legs proximal of the fracture site with respect to trabecular parameters, bone mass and connectivity density. Notably, structural parameters are altered in fractured legs when related to unfractured legs in WT but not in mice without SP and SNF.
The absence of SP and SNF reduces pain sensitivity and mechanical stability of bone in general. The micro-architecture of bone is profoundly impaired in the absence of intact SNF with a less drastic effect in SP-deficient mice. Both sympathetic and sensory neurotransmitters are indispensable for proper callus differentiation. Importantly, absence of SP reduces bone formation rate whereas absence of SNF induces bone resorption rate{maybe this could be beneficial as bone resorption would leave room for neo-growth plates}. Notably, fracture chondrocytes produce SP and its receptor NK1 and are positive for α-adrenoceptors indicating an endogenous callus signaling loop. We propose that sensory and sympathetic neurotransmitters have crucial trophic effects which are essential for proper bone formation in addition to their classical neurological actions.”

“Under rigid, stable fixation regimen, bone regenerates with no or only minor callus formation”

“When applying more flexible fixation regimens, bone healing occurs in consecutive stages which involve intense callus formation. Firstly, an acute inflammatory response and recruitment of mesenchymal stem cells (mesenchymal callus) occur in order to subsequently generate a primary cartilaginous callus populated mostly with chondrocytes (soft callus). Later, this cartilaginous callus undergoes revascularization and calcification (calcified hard callus) and is finally remodeled to fully restore a normal bony structure and architecture”<-The idea with LSJL is that perhaps the accute inflammatory respose and recruitment of mesenchymal stem cells can occur without a fracture.

“SP plays a role in pain transmission; tibial fractures cause an early and strong induction of sensory nerve regeneration and growth into the site of injury (sensory sprouting). The presence of NK1 receptors was demonstrated on bone cells”  SP can also affect proliferation in mesenchymal stem cells.

“skeletal growth or activity of bone tissue might be regulated by SNF”

“At day 5 after fracture, when chondrogenic differentiation starts, a substantial number of mesenchymal and chondrocyte-like cells stained positive for NK1R and some cells double-stained for SP. At day 9 after fracture, when most of the callus matrix has adopted a cartilaginous phenotype (soft callus), nearly all of the callus chondrocytes were SP- and NK1R-positive. At 13 days after fracture, when remodeling of the callus progressed toward tissue mineralization and the bony, hard callus was about to be formed, number of SP-positive callus cells appeared to be reduced compared to day 9 but NK1R staining seems to be unaltered in hypertrophic chondrocytes. SP- and NK1R staining pattern in sympathectomized mice was similar to WT”

“mesenchymal callus cells and periosteum stained positive for α1d adrenergic receptor 5 days after fracture whereas only few chondrocyte-like cells were α1d-positive”

Growth plate cellular senescence preceeds epiphyseal fusion.  It is the reason why people stop growing.  If we stop cellular senescence we can keep growing.  And this study states that stem cell growth is not based on demand.  Demand for stem cells too high to be kept up with may lead to cancer growth.  However, supplements, reduction of environmental stressors, and mechanical loading may be some ways to stimulate stem cell health and renewal.  The key to LSJL success on restoring growth renewal may be based on it’s ability to stimulate stem cell proliferation and impair cellular senescence.  As this study suggests that microcracks that occur during old age increase the demand for stem cells but the body doesn’t generate new stem cells or increase proliferation to compensate.  However, unlike microcracks due to degradation LSJL places forces directly on the cells themselves.  I will be posting some updates on the LSJL method as soon as the comments section is up(after Michael has resolved some legal issues).

Age-specific bone tumour incidence rates are governed by stem cell exhaustion influencing the supply and demand of progenitor cells

“Knudson’s carcinogenic model, which simulates incidence rates for retinoblastoma, provides compelling evidence for a two-stage mutational process. However, for more complex cancers, existing multistage models are less convincing. To fill this gap, I hypothesize that neoplasms preferentially arise when stem cell exhaustion creates a short supply of progenitor cells at ages of high proliferative demand. To test this hypothesis, published datasets were employed to model the age distribution of osteochondroma, a benign lesion, and osteosarcoma, a malignant one. The supply of chondrogenic stem-like cells in femur growth plates of children and adolescents was evaluated and compared with the progenitor cell demand of longitudinal bone growth. Similarly, the supply of osteoprogenitor cells from birth to old age was compared with the demands of bone formation. Progenitor cell demand-to-supply ratios are a good risk indicator, exhibiting similar trends to the unimodal and bimodal age distributions of osteochondroma and osteosarcoma, respectively. The hypothesis also helps explain Peto’s paradox and the finding that taller individuals are more prone to cancers and have shorter lifespans. The hypothesis was tested, in the manner of Knudson, by its ability to convincingly explain and demonstrate, for the first time, a bone tumour’s bimodal age-incidence curve.”

“Osteochondroma is the most common benign bone tumour, occurring as an abnormal osteocartilaginous outgrowth of the epiphyseal growth plate with a low rate (≤2%) of malignant transformation to secondary chondrosarcoma”

“In adolescents, growth plate senescence is followed by epiphyseal fusion when osteochondroma stop growing.”

“Osteosarcoma is the most common primary bone malignancy (excluding multiple myeloma), originating from the transformation of aberrant bone-forming mesenchymal stem cells (MSC), also known as marrow stromal cells”

“Differentiation is impaired by replicative senescence”

“Post-natal stem cell replication leads to telomere shortening in somatic tissues, as telomerase activity in humans is not at sustaining levels. At the Hayflick limit of cell division, telomeres reach a critical length, triggering cells to become senescent or apoptotic. A recent publication provides quantitative evidence that soft tissue organ mass loss in humans aged 25–70 is significantly associated with the log of shorter cell turnover times, implicating stem cell exhaustion and replicative senescence in normal ageing”

“MSC, like other stem cells, have been observed to have a self-renewal and proliferative capacity that diminishes with age, and that is regulated by different pathways, such as antioxidant defence, DNA repair, and protein turnover, before entering a senescence-associated proliferation arrest”

“There is a normal trade-off for a stem cell to self-renew or produce differentiated and differentiating progeny. High stem cell demand relative to supply appears to produce stem cell progeny with stopped differentiation but with self-renewal properties, perhaps triggered by senescent cell secretions”

Here’s a graph describing the stem cell pool:

“A good marker of the skeletal demand for osteoprogenitors involved in bone formation (as opposed to osteoclast bone resorption activity) is serum BALP activity. This marker is particularly sensitive to physiological changes such as adolescent growth spurt and menopause. The demand for progenitor cells participating in bone formation, as measured by BALP activity, is highest in newborns, slowly declines until early adolescence in girls and late adolescence in boys, then decreases rapidly to its lowest level in young adults, before increasing again in the latter half of adulthood to cope with the decline in osteocyte density and repair of accumulated damage related to ageing, e.g. bone microcracks”

“senescent cells can arise due to “epigenetic senescence” (initiated by histones altering activity of genes), stress-induced senescence or replicative senescence”<-Stress-induced sensescence is the easiest to avoid and epigenetic senescence can be affected by supplements.  Replicative senescence may be influenced by mechanical loading.

“cell proliferation is not in itself a risk factor for tumogenesis; instead, neoplastic promotion or progression occurs preferentially when, for example, stem cell exhaustion creates a short supply of progenitor cells at ages of high mitogenic demand.”

” Ionizing radiation exposures produce free radicals and reactive oxygen species that excite a high proliferative demand on MSC involved in inflammation and subsequent fibrosis. When bone marrow is subjected to a high dose, there is an increase in expression of the ageing and senescent cell biomarker p16INK4a reducing the capacity for stem cell renewal and, consequently, causing a decline in cellularity commensurate with premature ageing”

Predicting and Validating the Pathway of Wnt3a-Driven Suppression of Osteoclastogenesis.

“we examined Wnt3a-driven regulation of osteoclast development. Mouse bone marrow-derived cells were incubated with RANKL in the presence and absence of Wnt3a. Using microarray mRNA expression data, we conducted a principal component analysis and predicted transcription factor binding sites (TFBS) that were potentially involved in the responses to RANKL and Wnt3a. The principal component analysis predicted potential Wnt3a responsive regulators that would reverse osteoclast development, and a TFBS prediction algorithm indicated that the AP1 binding site would be linked to Wnt3a-driven suppression. Since c-Fos was upregulated by RANKL and downregulated by Wnt3a in a dose-dependent manner, we examined its role using RNA interference. The partial silencing of c-Fos suppressed RANKL-driven osteoclastogenesis by downregulating NFATc1, a master transcription factor of osteoclast development. Although the involvement of c-Myc was predicted and partial silencing c-Myc slightly reduced the level of TRAP, c-Myc silencing did not alter expression of NFATc1. Collectively, the presented systems-biology approach demonstrates that Wnt3a attenuates RANKL-driven osteoclastogenesis by blocking c-Fos expression and suggests that mechanotransduction of bone alters the development of not only osteoblasts but also osteoclasts through Wnt signaling.”

LSJL upregulates c-Fos.  In all likelihood LSJL alters Wnt3a expression but there wasn’t any evidence in the LSJL gene expression study.  Fluid flow upregulates Wnt3a and LSJL does involve fluid flow although this was only in osteocytes and we’d want to know the effects on stem cells or chondrocytes for height growth purposes.  No evidence that LSJL alters Nfatc1 expression but it likely does and Salubrinal alters Nfatc1 expression.  According to a diagram, from that study LSJL would increase Nfactc1 expression but it reduces levels of phosphorylated eif2a.

“Wnt5a activates noncanonical Wnt signaling through a receptor tyrosine kinase-like orphan receptor and stimulates osteoclastogenesis. Wnt10b is required for maintenance of mesenchymal progenitors, and its deficiency leads to loss of bone mass. Wnt14 enhances endochondral ossification and accelerates chondrocyte maturation”<-Wnt14 may alter height.  However, Wnt14 does suppress chondrogenic genes and I couldn’t find any studies stating that Wnt14 transgenes or knockout causes overgrowth or undergrowth.  Genetic association study of WNT10B polymorphisms with BMD and adiposity parameters in Danish and Belgian males., says that Wnt10b may have an effect on height but more testing needs to be done.

“Mouse bone marrow cells isolated from long bones (femur and tibia) as well as RAW264.7 mouse pre-osteoclast cells [were used in the study]”.  If mouse bone marrow cells were used it may have ramifications for how stem cells are affected by stimuli and finding the right stimuli is the key for height growth.

“Administration of RANKL to bone marrow cells significantly increased the number of TRAP-positive multi-nucleated cells{osteoclast cells are trap-positive}. In response to 100 or 200 ng/ml of Wnt3a, the number of TRAP-positive cells was reduced in a dose-dependent manner. The observed suppression of osteoclast development by Wnt3a was associated with a decrease in the phosphorylated form of β-catenin (p-β-catenin) as well as NFATc1 ”

“Wnt3a-induced reduction of the relative mRNA expression levels of the genes (NFATc1, TRAP, OSCAR, MMP9, and cathepsin K) linked to osteoclastogenesis on days 1 and 2 in bone marrow cells”<-Note that none of these levels were lower than control(The cells that were not exposed to RANKL) and the reduction was dose dependent until at least 200ng/ml.

Wnt3a downregulates C-Fos and C-Fos may be an important part in LSJL induced growth.

Wnt3a upregulated Egr1, Notch1, and Tgif1 at greater levels than control so excess levels of Wnt3a may have an effect on stimulating on those genes even without altering cells exposed to RANKL.  It downregulated Hmga1, Smad3, Dnmt3a, and Bach1 versus control.  Smad3 is involved in TGF-Beta induced chondrogenesis.  Dnmt3a promotes DNA methylation.

RECENT RESEARCH ON THE GROWTH PLATE: Mechanisms for growth plate injury repair and potential cell-based therapies for regeneration

“[The growth plate] functions to produce a mineralised cartilaginous scaffold to which new trabecular bone is formed via a tightly controlled two-step process (called endochondral ossification) involving chondrogenesis and osteogenesis ”

“The resting zone has previously been thought to play a very minimal role during endochondral ossification as the pre-chondrocytes/cells within this zone proliferate minimally. However, studies have indicated the importance of the resting zone as it acts as a reservoir of stem cells/pre-chondrocytes for the chondrocytes in the adjacent proliferative zone”

“The proliferative zone is responsible for matrix production (including collagen-2 and aggrecan) and cellular division during endochondral ossification. The height of the proliferative zone directly correlates with the extent of longitudinal growth that can be achieved by the long bone. As regulated by various signalling pathways including parathyroid hormone-related protein, insulin-like growth factor (IGF1), bone morphogenic protein (BMP), Wnt/B-catenin, fibroblast growth factor (FGF) and others , chondrocytes cease to proliferate and become hypertrophic. The hypertrophic chondrocytes produce collagen-10 which is involved with matrix mineralisation. Together with the action of angiogenic factor vascular endothelial growth factor (VEGF) produced by hypertrophic chondrocytes and a low oxygen tension, the lower hypertrophic zone attracts blood vessel invasion from the adjacent metaphyseal bone, which brings along mineralised cartilage-resorptive cells (chondroclasts), bone-forming cells (osteoblasts) and bone-resorptive cells (osteoclasts) to convert the mineralised cartilage scaffold into trabecular bone in metaphysis.”

“The initial inflammatory response involves an influx of key inflammatory cells into the growth plate injury site and up-regulation of inflammatory cytokines/mediators and some growth factors (A). The fibrogenic phase involves an influx of fibrogenic and progenitor cells containing MSC-like cells (B). The osteogenic phase involves the osteogenic and chondrogenic differentiation, formation of bony trabeculae together with angiogenesis within the injury site (C). The remodelling phase involves the maturation and active remodelling of the newly formed bony trabeculae as well as disappearance of cartilaginous repair tissue (D).”<-Cinc1 is also known as IL8 or CXCL1{Which is upregulated by LSJL}

“neutrophil-mediated inflammatory response was found to modulate downstream injury repair events. Following the depletion of neutrophils with a neutralising antibody, an increase in the undesirable bony repair tissue [occurred] with increased expression in bone-related genes such as Runx2 and osteocalcin, but decreased expression in cartilage-related genes Sox9 and collagen-2 ”

“Blocking TNFa resulted in a clear delay in the subsequent mesenchymal infiltration response and a reduction of the proliferation of these cells”

“At the growth plate injury site, some of these cells were found to express growth factors including BMPs, platelet-derived growth factor (PDGF) and FGF2 and receptors for BMPs and PDGF. In addition, some of these cells were found to be MSC like as they expressed the stem cell marker alpha-smooth muscle actin{acta2 which is upregulated by LSJL}”

“this influx of mesenchymal cells may contain a myriad of cells including MSC-like cells, osteoprogenitor cells, pre-osteoblasts, and/or pre-chondroblasts (either pre-existing or newly derived from the infiltrated MSCs).”

“significant peak in the mRNA expression of platelet-derived growth factor (Pdgf) and fibroblast growth factor 2 (Fgf2){up} following the initial inflammatory phase, suggesting a potential regulatory role for these two growth factors during this phase”

“In rats with growth plate injury, the inhibition of PDGF signalling caused a significant reduction in the amount of mesenchymal infiltrate, decreased amounts of bony and/or cartilage repair tissues, and thus an overall delay in bony repair 14 days post-injury”

“At the injured growth plate, bone formation has been observed to commence around day 7 with the appearance of bony trabeculae, and bone remodelling has been observed by day 14 with the appearance of bone marrow cells in between bony trabeculae”

“During the osteogenic phase of the growth plate injury repair process, the cells within the fibrogenic infiltrate differentiate into Runx2 and alkaline phosphatase-immunopositive osteoblasts and produce increased levels of bone matrix protein osteocalcin (both mRNA and protein) during days 8–14”

“after a ‘fibrous tissue’ is formed from the infiltrated stromal cells at an injured growth plate, its invasion by new blood vessels is a prerequisite for its osseous transformation”

“the absence of VEGF delayed bone formation by halting the initial soft callus from being converted into hard bony callus. ”

“osterix over-expression can induce bone healing”

“PKD up-regulates osterix and [is] important for osteoblast differentiation. Inhibition of PKD suppressed bony repair but induced more chondrogenic differentiation at the injury site”

“over-expression of osterix in osteochondroprogenitor cells resulted in a decrease in chondrogenic transcription factor Sox9.”

“In a rat tibial drill hole growth plate injury model, levels of Bmp2 mRNA expression were found notably increased in the early part of the fibrogenic phase and then again later during the osteogenic phase”

“synovium-derived MSCs in particular had the greatest capability to enhance the chondrogenic differentiation potential when compared with any other mesenchymal tissue-derived cells. However, other studies have reported that bone marrow-derived MSCs (BMMSCs) are most suitable for cartilage tissue engineering, as they possess higher proliferation rates and higher levels of expression of cartilage-specific genes, when compared with MSCs derived from other tissues”

“MSCs were successfully isolated directly from murine epiphysis.  This novel type of MSCs could potentially be better than BMMSCs as they have shown greater capacities in growth and differentiation potential as well as possessing immunosuppressive and anti-inflammatory properties”